(1) Field of the Invention
The present invention generally relates to novel modified hemoglobin and novel methods for modifying hemoglobin. More specifically, the invention relates to novel hemoglobin compositions comprising polyalkylene glycols and methods for making those hemoglobin compositions.
(2) Description of the Related Art
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Hemoglobin (Hb) is the major constituent of the erythrocyte which carries oxygen from the lungs throughout the body. When contained in red blood cells, Hb exists as a tetramer structure composed of two oxygen linked αβ dimers, each having a molecular weight of about 32 Kd. Each α and β subunit of each dimer has a protein chain and a heme molecule.
The sequences of the α and β protein chains are known. Hb is a potentially useful blood substitute for transfusions, and has been proposed as a reagent to trap nitric oxide in septic shocks, and to modulate tissue oxygenation during radiation therapy of cancer. Recombinant DNA technology also has afforded the generation of modified Hb with oxygen affinities modulated for special needs of individual therapeutic applications.
The potential use of Hb as blood substitutes in transfusions or other therapeutic applications, however, has been hampered by the short circulation half-life of Hb. In solution outside of the red blood cell, Hb readily dissociates from its tetrameric form into its dimers and even monomers, which are rapidly filtered through the kidneys. Accordingly, a multitude of methods for crossbridging Hb (e.g. bifunctional modification) and other means for increasing the hydrodynamic volume of Hb (e.g. monofunctional decoration) have been devised to limit or prevent the extravasation of Hb.
Simon and Konigsberg (1966) reports the use of bis(N-maleimidomethyl) ether (BME) to generate intramolecularly crosslinked Hb. Bunn et al. (1969) later reported that BME crosslinked Hb increased the half-life of Hb four-fold when infused into rats and dogs. However, the crosslinking of Hb with BME resulted in a concomitant increase in the oxygen affinity of Hb which prevented its use as a potential Hb-based oxygen carrier.
Xue and Wong (1994) describes many of the current methods for crosslinking Hb. These include the use of dextran, hydroxyethyl starch, inulin, polyvinylpyrrolidone, and polyethylene glycol as crosslinkers for Hb. Other crosslinkers include glycoaldehyde and glutaraldehyde (MacDonald and Pepper, 1994); bis(3,5-dibromosalicyl) fumarate (Walder, R. Y., et al., 1994); acyl phosphate esters (Kluger et al., 1994 U.S. Pat. No. 5,334,707); bissulfosuccinimidyl esters of aliphatic dicarboxylic acids (Manjula et al., 1994); and benzenepentacarboxylate (U.S. Pat. No. 5,349,054).
Nho et al. (1994) describes the monofunctional decoration of hemoglobin with polyethylene glycol. Similar modification of the hemoglobin molecule are described in U.S. Pat. Nos. 4,301,144; 4,412,989; 4,670,417; 5,234,903; and 5,312,808, and WIPO publication WO 94/04193.
The various modified hemoglobins described in the references cited above can be broadly grouped into three classes. (i) intramolecularly crossbridged Hb tetramers, (ii) inter and intra molecularly cross bridged Hb polymers, and (iii) Hb surface decorated with inert polymers such as polyethylene glycol (PEG). All these three classes of modified hemoglobins prevent glomerular filtration of acellular Hb and hence do not exhibit any nephrotoxicity that is associated with unmodified acellular Hb. However, the intrinsic propensity of Hb to bind nitric oxide, and hence its ability to influence vascular tone when present as an acellular component has been an obstacle to the widespread adoption of acellular Hb for therapeutic purposes. The different classes of modified Hb exhibit different degrees of vasoactivity. The intramolecularly crossbridged Hb, with its molecular size of 64,000 daltons, exhibited the highest vasoactivity, which is comparable to that of unmodified Hb. The intra and intermolecularly crossbridged species of Hb, with apparent molecular size of 200,000 to 300,000 exhibit a somewhat lowered vasoactivity relative to the parent Hb (or intramolecularly crossbridged HbA). The samples of Hb surface decorated with PEG chains with an apparent molecular size of 275,000 daltons or higher do not exhibit any vasoactivity. Thus, the increased molecular size of Hb appears to have reduced the vasoactivity of the product, presumably minimizing the extravasation of the sample into the interstitial space. This observation has presented a new approach to overcome the vasoactivity mediated toxicity of acellular Hb, distinct from approaches under development to engineer the Hb molecule through site directed mutagenesis to suppress the affinity of heme towards nitric oxide. The higher viscosity and the colloidal osmotic pressure of the solutions of surface decorated Hb appears to have other beneficial effects as well (Vandegriff et al., 1997; Winslow et al., 1998).
The surface decorated Hb investigated to date carry about ten PEG-5000 chains per tetramer (total mass of about 50,000 daltons of PEG per tetramer). The PEG-chains in this sample are linked to the surface α and/or ε-amino groups of Hb through isopeptide linkage (succinimidyl chemistry based PEGalation). Such preparations of surface decorated Hb do not increase the blood pressure, systemic vascular resistance remained unchanged, and tissue oxygenation are maintained at a level comparable to that of blood, even though the oxygen affinity of these preparations are higher than that of erythrocytes (Winslow et al., 1998).
One of the limitations of the above-described succinimidyl chemistry for surface decoration of Hb with PEG chains at the amino groups of Hb, is that the isopeptide linkage generated between Hb and the PEG-molecule does not carry the original positive charge of the amino (α or ε) of Hb. To overcome this limitation of earlier surface decoration chemistry, a novel protocol was recently developed to attach PEG-chains to Hb using the ε-amino groups of its surface Lys residues wherein the Hb still retains the original positive charge of the amino groups (U.S. Pat. No. 5,585,484). This involves amidination of the ε-amino groups of Hb by iminothiolane to introduce sulfhydryl groups on to the protein, which are subsequently targeted as the modification sites for PEGalation using maleimide chemistry-based PEG reagents. This approach has at least two additional specific advantages over the previously used succinimidyl chemistry: (1) the very high reactivity and selectivity of the maleimide based reagents to the sulfhydryl groups facilitates the near quantitative modification of the thiols with a limited excess of the reagents (in this case maleidophenyl PEG-chains), and (2) the thiol group of iminothiolane is latent and is generated only in situ as a consequence of the reaction of the reagent with the protein amino groups. Accordingly, Hb can be incubated simultaneously with the thiolating and the PEGalating reagents for surface decoration with PEG-chains.
Despite its advantages, the thiolation mediated, maleimide chemistry based surface decoration procedure described in U.S. Pat. No. 5,585,484 results in the modification of Cys-93(β) by the maleidophenyl PEG. This modification leads to an increase in the oxygen affinity of Hb.
Most of the known compounds used to modify Hb are difficult to synthesize, do not modify Hb in an efficient manner, cannot be manipulated quantitatively to form the desired modification, and/or lower or raise the oxygen affinity of the modified hemoglobin. Accordingly, there exists a need for new synthetic compounds, and new methods which can modify hemoglobin in an efficient and focused manner, and do not substantially affect the oxygen affinity of the modified hemoglobin. The present invention satisfies this need.